Clostridium beijerinckii NRRL B-598 is a sporulating, butanol and hydrogen producing strain that utilizes carbohydrates by the acetone-butanol-ethanol (ABE) fermentative pathway. The pathway consists of two metabolic phases, acidogenesis and solventogenesis, from which the latter one can be coupled with sporulation. Thorough transcriptomic profiling during a complete life cycle and both metabolic phases completed with flow cytometry, microscopy and a metabolites analysis helped to find out key genes involved in particular cellular events. The description of genes/operons that are closely involved in metabolism or the cell cycle is a necessary condition for metabolic engineering of the strain and will be valuable for all C. beijerinckii strains and other Clostridial species. The study focused on glucose transport and catabolism, hydrogen formation, metabolic stress response, binary fission, motility/chemotaxis and sporulation, which resulted in the composition of the unique image reflecting clostridial population changes. Surprisingly, the main change in expression of individual genes was coupled with the sporulation start and not with the transition from acidogenic to solventogenic metabolism. As expected, solvents formation started at pH decrease and the accumulation of butyric and acetic acids in the cultivation medium.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Clostridium beijerinckii cytologie genetika MeSH
- fermentace genetika MeSH
- fyziologický stres * genetika MeSH
- glukosa metabolismus MeSH
- kyseliny metabolismus MeSH
- mastné kyseliny metabolismus MeSH
- proteiny tepelného šoku genetika metabolismus MeSH
- regulace genové exprese u bakterií * MeSH
- rozpouštědla metabolismus MeSH
- spory bakteriální metabolismus MeSH
- transkriptom genetika MeSH
- vodík metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Zygosaccharomyces rouxii is a fructophilic yeast that consumes fructose preferably to glucose. This behavior seems to be related to sugar uptake. In this study, we constructed Z. rouxii single-, double-, and triple-deletion mutants in the UL4 strain background (a ura3 strain derived from CBS 732(T)) by deleting the genes encoding the specific fructose facilitator Z. rouxii Ffz1 (ZrFfz1), the fructose/glucose facilitator ZrFfz2, and/or the fructose symporter ZrFsy1. We analyzed the effects on the growth phenotype, on kinetic parameters of fructose and glucose uptake, and on sugar consumption profiles. No growth phenotype was observed on fructose or glucose upon deletion of FFZ genes. Deletion of ZrFFZ1 drastically reduced fructose transport capacity, increased glucose transport capacity, and eliminated the fructophilic character, while deletion of ZrFFZ2 had almost no effect. The strain in which both FFZ genes were deleted presented even higher consumption of glucose than strain Zrffz1Δ, probably due to a reduced repressing effect of fructose. This study confirms the molecular basis of the Z. rouxii fructophilic character, demonstrating that ZrFfz1 is essential for Z. rouxii fructophilic behavior. The gene is a good candidate to improve the fructose fermentation performance of industrial Saccharomyces cerevisiae strains.
- MeSH
- biologický transport genetika MeSH
- fermentace genetika MeSH
- fruktosa metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- genový knockdown MeSH
- glukosa metabolismus MeSH
- proliferace buněk genetika MeSH
- regulace genové exprese u hub MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Zygosaccharomyces genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Expresní systém kvasinky Pichia pastoris se ve velkém měřítku používá na produkci rekombinantních proteinů již několik desetiletí. Pichia pastoris v sobě spojuje výhody eukaryotních i prokaryotních expresních systému. V rámci této práce byly v bioreaktoru produkovány mutantní formy proteinu cryptogeinu. Bylo vyprodukováno a purifikováno pět proteinů: X24, L41F, V84F, V84F/L41F a K13V s průměrnými výtěžky 40-60 mg proteinu na litr bazálního media. Buněčné hustoty na konci fermentace dosahovali 250-400 g/l.
Expression system Pichia pastoris has been used for decades to production of recombinant proteins in large scale. Pichia pastoris combines the advantages of eukaryotic and prokaryotic expression systems. In this work mutant forms of protein cryptogein were produced in the fermenter. Five proteins: X24, L41F, V84F, V84F/L41F and K13V were produced in the bioreactor and purified with average yields of 40-60 mg protein per liter of basal media. Cell density was reached 250-400 g/l at the end of fermentation.
- Klíčová slova
- růstová křivka, fermentor,
- MeSH
- bioreaktory MeSH
- fermentace * fyziologie genetika MeSH
- fungální proteiny * biosyntéza genetika MeSH
- kultivační média * MeSH
- mikrobiologické techniky MeSH
- Pichia genetika metabolismus růst a vývoj MeSH
- proteomika * metody MeSH
- rekombinantní proteiny * biosyntéza genetika MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- alkoholické nápoje metody MeSH
- amylasy genetika MeSH
- exprese genu MeSH
- fermentace genetika MeSH
- kvasinky fyziologie MeSH
- Publikační typ
- srovnávací studie MeSH
